SUMMARY Current tools for rapidly characterizing the structures, heterogeneity, quality, and similarity of biomolecules do not provide the level of selectivity needed for many applications, including biotherapeutic development, production, and regulation. Mass spectrometry (MS) and ion mobility (IM) are particularly appealing for addressing these unmet needs due to their speed, sensitivity, tolerance for sample heterogeneity, and scalability. This project will result in a new platform that will enable higher-performance and higher-dimensional characterization of biomolecules, including intact proteins and multiprotein complexes. The new measurements enabled by this platform are not possible using existing IM-MS platforms. This platform will use modular components to separate, select, and trap native-like ions, which will be combined into a large array of modules (Aim 1) and integrated with complementary probes of ion structure (Aim 2). This platform will enable multiple dimensions of analysis that probe the shapes, stabilities, internal interactions, and reactivity of the ions. The orthogonality between these dimensions of analysis will increase the selectivity of measurements, without compromising the underlying strengths of IM and MS. These aims will be (Aim 3) evaluated using model proteins, protein-ligand complexes, and protein-protein complexes in order to assess the potential of this platform to answer challenging problems in biomedical research. More generally, this research will improve the feasibility of higher-dimensional IM-MS experiments and will improve the understanding of the orthogonality than can be achieved between each dimensions of analysis. This project benefits greatly from modular components and rapid prototyping, but with the outcomes of this research, we anticipate that this flexibility of experimental design and the selectivity of these measurements can be translated to architectures that are more user friendly (albeit less modular) and thereby have broad impact across the biomedical sciences and biopharmaceutical industries.